The present invention relates to the immobilization of specific binding ligands, such as nucleic acids and other ligands, in a known spatial arrangement. In another aspect, the invention relates to solid supports, such as oligonucleotide chips, incorporating such nucleic acids. In yet another aspect, the invention relates to photoreactive groups, to molecules and/or surfaces derivatized with such groups, and to the attachment of such molecules to support surfaces by the activation of such groups.
The development of oligonucleotide probe arrays, more commonly known as xe2x80x9cDNA chipsxe2x80x9d and GENE CHIP (a registered trademark of Affymetrix, Inc.), has made significant advances over the past few years, and is becoming the center of ever-increasing attention and heightened significance. See, for instance, Stipp, D., Fortune, p.56, Mar. 31, 1997. See also Borman, S., CandEN, p.42, Dec. 9, 1996, and Travis, J., Science News 151:144-145 (1997). These 2- or 3-cm square chips are capable of containing tens of thousands to hundreds of thousands of immobilized oligonucleotides, allowing researchers to witness for the first time the behavior of thousands of genes acting in concert. DNA chips are useful for observing unique gene expression patterns, gauging the success of drug treatment, tailoring medications to patients based upon their genetic makeup, sequencing genes, and conducting research in the area of genetic medicine. See also, xe2x80x9cMicrochip Arrays Put DNA on the Spotxe2x80x9d, R. Service, Science 282(5388):396-399, Oct. 16, 1998; and xe2x80x9cFomenting a Revolution, in Miniaturexe2x80x9d, I. Amato, Science 282(5388): 402-405, Oct. 16, 1998.
Typically, oligonucleotide probe arrays display specific oligonucleotide sequences at precise locations in an information rich format. In use, the hybridization pattern of a fluorescently labeled nucleic acid target is used to gain primary structure information for the target. This format can be applied to a broad range of nucleic acid sequence analysis problems including pathogen identification, forensic applications, monitoring mRNA expression and de novo sequencing. See, for instance, Lipshutz, R. J., et al., BioTechniques 19(3):442-447 (1995). Such arrays sometimes need to carry several tens of thousands, or even hundreds of thousands of individual probes. The chips also need to provide a broad range of sensitivities in order to detect sequences that can be expressed at levels anywhere from 1 to 10,000 copies per cell.
A variety of approaches have been developed for the fabrication and/or use of oligonucleotide probe arrays. See, for instance, Weaver, et al. (WO 92/10092) which describes a synthetic strategy for the creation of large scale chemical diversity on a solid-phase support. The system employs solid-phase chemistry, photolabile protecting groups and photolithography to achieve light-directed, spatially addressable, parallel chemical synthesis. Using the proper sequence of masks and chemical stepwise reactions, a defined set of oligonucleotides can be constructed, each in a predefined position on the surface of the array.
Using this technology, Affymetrix, Inc. (Santa Clara, Calif.), has developed libraries of unimolecular, double-stranded oligonucleotides on a solid support. See, for instance, U.S. Pat. No. 5,770,722 which describes arrays containing oligonucleotides from 4 to 100 nucleotides in length. The arrays comprise a solid support, an optional spacer, a first oligonucleotide, a second oligonucleotide that is complementary to the first, and a flexible linker or probe. The libraries described are useful for screening for such receptors as proteins, RNA or other molecules which bind double-stranded DNA. Another array developed by Affymetrix is described in U.S. Pat. No. 5,837,832. This reference describes methods for making high-density arrays of oligonucleotide probes on silica chips. The oligonucleotide probes are 9 to 20 nucleotides in length and are synthesized directly on a solid support. The arrays comprise oligonucleotide probes that are complementary to a section of the reference sequence.
Synteni (Palo Alto, Calif.) produces arrays of cDNA by applying polylysine to glass slides, followed by printing cDNA onto the coated slides. The arrays are then exposed to UV light, in order to crosslink the DNA with the polylysine. Unreacted polylysine is then blocked by reaction with succinic anhydride. These arrays, called xe2x80x9cGene Expression Microarraysxe2x80x9d (GEM(trademark)) are used by labeling mRNA prepared from a normal cell with a fluorescent dye, then labeling mRNA from an abnormal cell with a fluorescent dye of a different color. These two labeled mRNA molecules are simultaneously applied to the microarray, where they competitively bind to the immobilized cDNA molecules. This two color coding technique is used to identify the differences in gene expression between two cell samples. (Heller, R. A., et al., Proc. Natl. Acad. Sci. USA, 94:2150-2155 (1997)).
At least one group, Cantor, et al. (U.S. Pat. No. 5,795,714), describes methods for replicating arrays of probes which are said to be useful for the large scale manufacture of diagnostic aids. The patent includes a method for replicating an array of single-stranded probes on a solid support comprising the steps of:
a) synthesizing an array of nucleic acids each comprising a non-variant sequence of length C at a 3xe2x80x2-terminus and a variable sequence of length R at a 5xe2x80x2-terminus;
b) fixing the array to a first solid support;
c) synthesizing a set of nucleic acids each comprising a sequence complementary to the non-variant sequence;
d) hybridizing the nucleic acids of the set to the array;
e) enzymatically extending the nucleic acids of the set using the variable sequences of the array as templates;
f) denaturing the set of extended nucleic acids; and
g) fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes.
On a separate subject, the assignee of the present invention has previously described a variety of applications for the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, and 5,714,360 and International Patent Application Nos. PCT/US96/08797 (Virus Inactivating Coatings), PCT/US96/07695 (Capillary Endothelialization), and PCT/US97/05344 (Chain Transfer Agents).
In spite of the various developments to date, there remains a need for methods and reagents that improve the immobilization of nucleic acids onto a variety of support materials, e.g., in order to form oligonucleotide probe arrays. What is clearly needed are new and improved methods and reagents for reproducibly preparing specific binding ligand (e.g., nucleic acid) arrays in a cost-effective and efficient manner, while maintaining an accurate, sensitive product.
The present invention, in one preferred embodiment, provides a system for producing substantially identical specific binding ligand (e.g., nucleic acid) probe arrays, for instance, by preparing and replicating an original xe2x80x9cmasterxe2x80x9d array and/or by providing a reusable assay array that is capable of being regenerated. The present approach can be contrasted with the traditional approach of separately and individually preparing each probe array anew, without the efficiency of replicating a master array or the flexibility of regenerating an array for subsequent use. The present method can be adapted for use with conventional arrays, in order to provide replicates thereof, but is preferably used with a master array (and other components) specifically designed for such purposes, in the manner described herein.
In such a preferred embodiment, the present invention provides a method and system for reproducibly preparing an assay array, the system comprising:
a) a master array comprising a support surface having a plurality of xe2x80x9caddressxe2x80x9d ligands (e.g., oligonucleotide sequences) immobilized thereon, for instance, directly or via respective linking agents, such as xe2x80x9cmicro-beadsxe2x80x9d or other suitable linkers, the ligands being immobilized in the form of a patterned, and optionally random, array;
b) a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising a core (e.g., molecular or solid) having (preferably independently) attached thereto: (i) at least one molecule of a first ligand (e.g., oligonucleotide) binding domain, comprising a ligand selected to bind in a complementary manner to a particular address ligand of the master array, (ii) at least one molecule of a second ligand (e.g., oligonueleotide) binding domain, comprising a ligand selected to bind in a complementary manner to a characteristic ligand of a target nucleic acid sequence (e.g., a gene and/or gene fragment), and (iii) at least one molecule of a third (and preferably non-oligonucleotide) ligand, selected from the group consisting of binding ligands and polymerizable groups (e.g., acrylic or vinyl groups); and
c) an assay array support comprising a support surface for the replicate array, e.g., coated with attachment sites for the third ligand (such as, immobilized molecules of a corresponding binding partner specific for the third ligand).
A corresponding preferred method of the invention, wherein the address and target ligands are both nucleic acids, preferably oligonucleotides, involves the steps of: (1) providing a master array as described above, (2) attaching the multi-ligand conjugates thereto, by allowing their respective oligonucleotide binding domains to hybridize to the complementary address sequences of the master array, (3) bringing the assay array support into sufficient proximity with the master array, under conditions suitable to permit the attached multi-ligand conjugates to attach to the assay array support (e.g., by binding between the third ligands and the corresponding attachment sites present upon the assay support surface), and (4) disassociating the hybridized complementary oligonucleotides under conditions suitable to permit the assay array support to be recovered and used. The resulting assay array comprises an assay array support having attached thereto a plurality of multi-ligand conjugates (preferably still having complementary target oligonucleotides thereon) present in a pattern established by the master array.
In an alternative preferred embodiment, where the conjugate includes a polymerizable group (e.g., in addition to the third binding ligand, or in place of the third binding ligand), the multi-ligand conjugates can be maintained in their oriented positions upon the master array surface by polymerizing those groups in situ, in order to form a polymeric backing sufficient to permit the resultant polymerized layer to be supported and used, e.g., transferred to an assay array support, while retaining the spatial arrangement established by the master array addresses.
Once transferred to an assay array support, the second oligonucleotide binding domains, in turn, can be used in a conventional manner to determine the presence (e.g., in absolute or relative amounts) of one or more target nucleic acids in a sample. The order and arrangement of the second binding domains is predetermined and maintained in the course of the replicating method set forth herein. For instance, the resultant assay array can be used in a conventional manner, e.g., by contacting the array with a sample suspected of containing the target nucleic acid, under conditions suitable to permit any target nucleic acid to be hybridized and detected.
In other aspects, the invention provides a method of using such a system; the various components for use in such a system, including a kit or combination of one or more components; as well as an assay array formed by the method of the invention.